Sickle cell dehydration plays an important role in the pathogenesis of sickle cell disease. Due to the marked dependence of Hb S polymerization kinetic on Hb S concentration, Hb S polymerization and sickling are markedly increased in dehydrated erythrocytes. Identification of the prominent role of Gardos channel and K-Cl cotransport in sickle cell dehydration has made possible the development of specific therapies to prevent dehydration in vivo (e.g. increasing red cell Mg). However, much remains unknown concerning the regulatory properties of the K-Cl cotransport, both in vitro and in vivo, especially in relation to the hypoxic and pro-oxidant environment characteristic of sickle cell disease and Mg supplementation therapy. We propose to: 1) Characterize the regulation of human and mouse K-Cl cotransport by kinases and phosphatases: We propose in vitro experiments on normal and sickle (mouse and human) erythrocytes to study the role played by protein kinases and phosphatases in regulating K-Cl cotransport function, and determine the mechanism(s) responsible for K-Cl cotransport up-regulation in sickle cell disease. 2) Characterize how the interactions between cell Mg, urea, hypoxia and/or oxidative damage affect the functional properties of erythroid K-Cl cotransport: The effects of erythrocyte Mg on the modulation of K-Cl cotransport activity by hypoxia, urea, and oxidative damage will be studied in vitro. We will assess in transgenic sickle mice how Mg supplementation may modulate the effects of chronic hypoxia and of novel anti-oxidative agents. 3) Assess the effects of Mg supplementation on patients with SS disease: We propose a limited pilot study on 10 patients with SS disease to validate an oral formulation of Mg pidolate to be used for clinical studies in patients with sickle cell disease in the USA. This is a necessary preliminary step for moving to a large multi-center clinical study on the clinical benefits of Mg supplementation in sickle cell disease. These studies will provide a systematic characterization of the interactions between Mg, urea, hypoxia,oxidation and antioxidants on the molecular mechanisms regulating the K-Cl cotransport system of human and mouse erythrocytes. These aims are consistent with the long-term goals of the applicant, who is focused on the study on membrane transport and volume regulation and the continued development of new therapies for sickle cell disease.